Microglia are the resident immune cells of the central nervous system (CNS). In the CNS, calcium signaling is an essential secondary messenger system in astrocytes and neurons. However, in microglia, calcium signaling is not completely understood. In this proposal, we investigate microglial calcium signaling in health and disease. In our first aim, we explore whether microglial calcium signaling occurs in the awake mouse and whether that signaling is neuronal activity dependent using in vivo two-photon microscopy. Activity-dependent calcium signaling in microglia would suggest that this cell type could critically sense and respond to neuronal changes. The ability to sense neuronal activity may be particularly important during epilepsy development. In our second aim, we explore whether and how microglial calcium activity changes in a model of epilepsy development. In aim 2, we use in vivo, longitudinal two-photon imaging to study changes in calcium signaling during epileptogenesis. We hypothesize that the purinergic receptor, P2Y6, is essential for microglial calcium signaling in both health and disease. Our training plan utilizes selective pharmacology and gene knockout strategies to understand the mechanisms of microglial calcium signaling. Our proposed research is an important first step in understanding a critical secondary messenger system and its role in microglial-neuron interactions. Successful completion of these aims will advance our understanding of the epileptogenic process (NINDS Benchmark II for Epilepsy Research). Research will be performed at the Mayo Clinic, a renowned center for translational research and clinical care. Project mentors are experts in microglial physiology/pathophysiology (Dr. Long-Jun Wu), and mechanisms of epileptogenesis (Dr. Greg Worrell).